The role of CD4+ T cells in tumor and chronic viral immune responses

Abstract Immunotherapies are mainly aimed to promote a CD8+ T cell response rather than a CD4+ T cell response as cytotoxic T lymphocytes (CTLs) can directly kill target cells. Recently, CD4+ T cells have received more attention due to their diverse roles in tumors and chronic viral infections. In antitumor and antichronic viral responses, CD4+ T cells relay help signals through dendritic cells to indirectly regulate CD8+ T cell response, interact with B cells or macrophages to indirectly modulate humoral immunity or macrophage polarization, and inhibit tumor blood vessel formation. Additionally, CD4+ T cells can also exhibit direct cytotoxicity toward target cells. However, regulatory T cells exhibit immunosuppression and CD4+ T cells become exhausted, which promote tumor progression and chronic viral persistence. Finally, we also outline immunotherapies based on CD4+ T cells, including adoptive cell transfer, vaccines, and immune checkpoint blockade. Overall, this review summarizes diverse roles of CD4+ T cells in the antitumor or protumor and chronic viral responses, and also highlights the immunotherapies based on CD4+ T cells, giving a better understanding of their roles in tumors and chronic viral infections.

become exhausted. 1,2The process usually occurs in a hierarchical manner, starting with a significant reduction in interleukin (IL)-2 (IL-2) expression, which is essential for T-cell survival and proliferation, thus making it difficult to maintain their activation state. 3,4Furthermore, tumor necrosis factor (TNF), interferon-γ (IFN-γ), chemokines, and granzymes produced by activated T cells decrease sharply resulting in a loss of antitumor and antichronic viral function. 5Eventually, if the strong antigen stimulation persists, the specific cytotoxic T lymphocytes (CTLs) tend to disappear, especially in the absence of help from CD4 + T cells. 4,6,7Along with "functional exhaustion," exhausted CTLs specifically coexpress high levels of inhibitory receptors, including programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte associated antigen-4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), T cell immunoglobulin and mucin domain-containing protein-3 (TIM-3), also known as immune checkpoints, which substantially affect the cytotoxic ability of CTLs and contribute to maintain their exhausted condition. 6,8,9n this case, recently, CD4 + T cells have received more attention.
Recently, CD4 + T cells have received increasing attention in tumors and chronic viral infections.According to previous studies, CD4 + T cells provide help through costimulatory molecules or cytokines to optimize the cytotoxicity and longevity of CD8 + T cells and regulate TME. 10 Moreover, CD4 + T cells can also modulate other subsets to indirectly improve antitumor and antichronic viral responses, such as the interaction of CD4 + T cells with B cells, 11 the regulation of macrophage polarization, 12 and the modulation of vessel system formation in tumor tissue. 13In addition, cytotoxic CD4 + T cells have been proven to directly induce tumor cell apoptosis, mediated by granzymes and perforin. 14However, they also help form an immunosuppressive environment, which can prevent a favorable outcome in antitumor immunity. 15n this review, we mainly focus on how CD4 + T cells provide help to CD8 + T cells through dendritic cells (DCs) and other subsets in antitumor and antichronic viral immunity, how the cytotoxic CD4 + T cells directly kill target cells, and the immunosuppressive microenvironment mediated by CD4 + T cells in protumor and prochronic viral immunity.Meanwhile, we have also summarized antitumor and antichronic viral immunotherapies based on CD4 + T cells.

A BRIEF DESCRIPTION OF CD4 + T CELL CLASSIFICATION
Similar to CD8 + T cells, the recognition between TCR and specific epitopes binding in MHC-II on the surface of antigen-presenting cells (APCs) is first required for the priming of CD4 + T cells.Whereafter, multiple transcriptional programs mediated by different cytokines initiate the differentiation from naïve T cells to other sub-populations in order to eliminate different pathogens.Depending on different phenotypes and functions, CD4 + T cells are mainly classified into helper T (Th) lymphocytes and regulatory T (Treg) lymphocytes.The Th cells provide help to enhance the immune effects of CD8 + T cells and B cells, while Treg cells exhibit an immunosuppressive effect.Th lymphocytes mainly include Th1, Th2, Th17, and Tfh, according to the specific expression of transcription factors and the different secreted cytokines (Figure 1).

Th1 cells
7][18][19] Especially, IL-12 has been shown to have the ability to switch the Th17 cells to Th1 cells. 20Additionally, the differentiated Th2 cells transferred to lymphocytic choriomeningitis virus (LCMV)-infected mice could acquire Th1 cell phenotypes, characterized by an increase in INF-γ and a decrease in IL-4. 21In addition to these main transcription factors, there are other transcriptional factors critical in the polarization of Th1 cells, such as interferon regulatory factor (IRF)1 at the downstream of INF-γ 22 and RUNX3, which could promote the expression of INF-γ and suppress the expression of IL-4. 23,24IFN-γ produced by Th1 cells, referred to as an important antitumor cytokine, can enhance the antitumor response through various ways, such as promoting the activation of CTLs, upregulating the expression of MHC molecules on DCs to improve the efficiency of antigen presentation, promoting the generation of M1-like macrophages, inhibiting the suppressive microenvironment, and mediating the formation of the early vascular system in tumor tissue. 25,26In addition, IFN-γ is associated with a positive prognosis in ICB therapies [27][28][29] and patients with nonresponsive melanoma exhibit a loss of IFN-γ signaling. 30Similarly, a group of efficiently responsive CD4 + ICOS + (inducible co-stimulator) T cells is identified as a phenotype of Th1 cells in bladder cancer patients who are all treated with Ipilimumab (anti-CTLA-4).This efficiently responsive sub-population upregulates IFN-γ expression under the help from ICOS pathway, 31 which is also demonstrated in patients with metastatic melanoma 32 and murine tumor models. 33Nevertheless, other studies have found that IFN-γ in TME may also promote the expression levels of inhibitory receptors, such as PD-1, PD-L1, LAG-3, and TIM-3, which have been proven to help tumor escape. 27,34While during antiviral infection, IFN-γ can induce IFN-stimulated genes (ISGs) 35,36 and promote IgG2a class switching, which subsequently exert antiviral effect. 37Additionally, the CD4 + T cell pools of HIV controllers are enriched in Th1 phenotype 38,39 and it is demonstrated that the decreased expression of ISG reduces the HIV sensitivity of Th1 cells. 40

Th2 cells
Th2 cells are generated from Th0 cells in the presence of IL-4 and characteristically express STAT6, which subsequently promotes the expression of GATA3 and inhibits the expression of Th1-related transcriptional factors STAT4 and T-bet.2][43][44] Previous research also demonstrated that Th2 cells could secrete IL-4 and IL-5 contributing to antitumor response by regulating TME and recruiting other effector cells in the tumor model with vaccination. 45Accordingly, in an IFN-γ-deficient murine model, the antitumor response significantly reduce in the absence of Th2 cytokines.Furthermore, tumor-infiltrating eosinophils decrease in IL-5deficient mice leading to a loss of antitumor response. 45onsistently, blocking IL-5 pathway also contributes to tumor growth, while overexpressing IL-5 or IL-4 helps to predominantly improve eosinophils, macrophages infiltration to enhance tumor clearance. 45,46In chimeric antigen receptor (CAR)-T therapy, it was found that efficiently responsive cells not only had Th1-associated phenotypes, but also could produce Th2-related cytokines such as IL-5 and IL-13. 47However, Th2 cells have been reported to exhibit a protumor effect in breast and cervical tumors. 48,491][52][53] In antiviral response, in addition to the positive effect induced by IL-4, Th2 cells also play negative roles in multiple viral infections, including influenza virus, 54,55 vaccinia virus, 56 respiratory syncytial virus (RSV), 57 and herpes simplex virus (HSV) infections. 58

Th9 cells
Th9 cells, which was initially considered as one of subpopulations in Th2 cells, have been distinguished due to the specific expression of IL-9 recently.The transforming growth factor (TGF)-β, IL-4, and the transcription factor IRF4 are required for the Th9 cells, which can be involved in response to parasitic infections and allergic reaction. 59nd in antitumor response, IL-9 promotes the representing ability of DCs 60 and mediates the mast cells to prevent tumor growth. 61,62Moreover, adoptive transfer Th9 cells exhibit a robust antitumor immunity against melanoma. 63n addition, glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) agonistic antibody DTA-1 can induce IL-9 to enhance the CTLs response by strengthening the function of DCs. 60

Th17 cells
5][66][67] In addition to the traditional related cytokines-TGF-β and IL-6, the combination of IL-23, IL-6, and IL-1β can also induce the production of IL-17 even in the absence of TGF-β. 65Th17 cells are considered to play a bifacial role in the response to extracellular infections and autoimmune diseases. 64,65,67In influenza and vaccinia viral infections, Th17 cells might contribute to immunopathology, 68,69 while they also exert protective functions by increasing CXC-chemokines induced by IL-17 to recruit neutrophils. 70Similarly, Th17 cells are also considered to play a bifacial role in antitumor immunity.][73][74] Furthermore, a previous study demonstrated that IL-17 could promote tumor progression by inducing the vascular system formation in tumor tissue. 75Additionally, Th17 cells have a positive role in antitumor immunity, which is mainly reflected in promoting the cell migration, such as NK cells and neutrophils, and regulating cell composition in the TME. 76Furthermore, Th17 cells can produce IL-21, which contributes to the generation and infiltration of CX3CR1 + CD8 + T cells to promote tumor clearance in the melanoma model. 77

Th22
Different from other Th cells, Th22 cells were distinguished by the secreting of IL-22, a member of IL-10 family, and without the production of IL-17, IL-4, and IFN-γ. 78,79In human lymphocytes, during the generation of Th22 cells, which were thought to be connected with skin immunity, 78,80 the transcriptional factor aryl hydrocarbon receptor has been proved to play a significant role in upregulating the production of IL-22 instead of IL-17. 78,81It has been demonstrated that Th22 cells and IL-22 were associated with the progress of various human cancers such as gastric cancer, 82 hepatocellular carcinoma, 83 colon cancer, 84 and lung cancer. 85During HIV infection, IL-22-produced cells appeared to sharply decrease, while the increase of IL-22 could be observed after long-term antiviral therapy. 86While in liver of mice and human with chronic hepatitis B viral infection, the expression of IL-22 was corelated with the grade of inflammation and proliferation of liver stem/progenitor cells. 87Despite of the secreting of IL-22, Th22 cells also express chemokine receptor C-C chemokine receptor (CCR)6, CCR4, and CCR10. 79

Tfh cells
Tfh cells with the production of IL-4 and IL-21 and the expression of Bcl-6 [88][89][90] and STAT3 mediated by IL-6 and IL-21 are supposed to help the B cell maturity and germinal center (GC) formation. 11,91,926][97] In addition, Tfh cells can express ICOS and CD40L, which contribute to the interaction with B cells and play a significant role in the antitumor immune response. 94,98The interaction of ICOS and ICOSL could activate the downstream P13K signaling, which is required for the differentiation of Tfh cells. 99Unlike other CD4 + T cells, Tfh cells can be distinguished by the expression of CXCR5, 100 which is indirectly maintained by the expression of Bcl-6. 101In murine tumor models, it was found that Tfh cells were closely linked with the recruitment of B cells and the formation of tertiary lymphoid structures (TLS), which subsequently affected the tumor control. 102,103In breast cancer patients, the expression levels of Tfh gene signatures were correlated with TLS activity and clinical outcomes. 104Although the previous studies on CD4 + T cells in chronic infections have concentrated on Th1 cells, it has been proven that Tfh cells also play an important role in antiviral infection. 105For example, in LCMV and vaccinia virus infection models, the absence of Tfh cells led to a decrease in both quantity and quality of antiviral antibodies. 106,107Likewise, the frequency of Tfh cells trends to increase after influenza vaccines injections in humans. 108,109

Treg cells
5][116][117] In addition to FOXP3, the expression of CD25 is also a biomarker in Treg cells.2][123] Moreover, the expression of suppressor of cytokine signaling 1 (SOCS1) in Treg cells can inhibit the production of IFN-γ and IL-17 to sustain Foxp3 expression mediated by inhibiting the STAT1 and STAT3 signal pathways. 124TGF-β and IL-10 secreted by Treg cells also contribute to mediating an immunosuppressive TME. 125,1267][128] Most studies on the impact of Treg cells in viral infections are based on chronic infections and the knowledge about their role in acute infections is not been well understood.It has been observed that the frequency of Treg cells increases in various human and animal viral infections.Accordingly, Treg cells can contribute to limiting the tissue immunoinflammatory damage, such as HSV-induced eye damage, and also induce immune dysfunction in HCV and HIV infections. 129

CD4 + T CELL ROLES IN ANTITUMOR AND ANTICHRONIC VIRAL RESPONSES
CD4 + T cells engage in antitumor and antichronic viral immune responses via several approaches, such as the indirect approaches and the direct approach.In indirect approaches, CD4 + T cells indirectly regulate CD8 + T cell response by DCs, interact with B cells or macrophages to indirectly modulate humoral immunity or macrophage polarization, and inhibit tumor blood vessel formation to promote antitumor response.Additionally, cytotoxic CD4 + T cells also can directly kill target cells to enhance antitumor immunity.

CD4
+ T cell help shape CD8 + T cell response via DCs CD8 + T cells activate and differentiate into cytotoxic CD8 + T cells after receiving MHC-I signaling on tumor cells or APCs and in this process, DCs play a crucial role in priming CD8 + T cells due to their strong antigen presentation abilities. 130,131DC subsets are categorized as plasmacytoid DCs, monocyte-derived DCs, and classical DCs (cDCs) based on their developmental process, phenotypes, and function.cDCs can be further divided into two major subsets including CD8α + and/or CD103 + cDC1s and CD11b + cDC2s, 132,133 and both of them contribute to the immune response against tumor cells.cDC2s prefer to activate CD4 + T cells, [134][135][136] whereas cDC1s mediate the priming of CD8 + T cells. 132,133,137revious studies have proposed a two-step model to understand the priming process of CD8 + T cells.9][140] In the next step, activated CD8 + T cells secrete XC-chemokine ligand 1 (XCL1), recruiting XC-chemokine receptor 1 XCR1 + cDC1s, which sequentially interact with CD4 + T cells and CD8 + T cells and transmit the help signals to CD8 + T cells, 139,140 while the assistance from CD4 + T cells is highlighted in the second step in which the same cDC1 is required to present the same epitopes to both CD4 + T and CD8 + T cells.Considering the fact that specific antigens derived from endocytosed proteins are presented by MHC-II on DCs after processing and also effectively presented by MHC-I as a consequence of a particular phenomenon called antigen cross-presentation in DCs 141 -originally found in Bevan's research and named "cross-priming" in 1976. 142During this time, DCs can relay help from CD4 + T cells to CD8 + T cells (Figure 2).4][145][146] Parallel results were detected in vaccine models, suggesting the necessity of CD4 + T cell help while using long peptides that needed to be processed. 147,148

DCs relay CD4 + T cell help through costimulatory molecules
The delivery of CD4 + T cell help in antigen crosspresentation mainly relies on the interaction between CD40L on activated CD4 + T cells and CD40 on DCs. 143tudies on therapeutic antitumor vaccines have provided favorable evidence that, in the absence of CD40 signal, vaccination with MHC-I-binding peptides fails to induce an effective CTL response, while the vaccination with F I G U R E 2 CD4 + T cells help CD8 + T cells to promote immune response by DCs.CD4 + T cells interact with cDC1s by costimulatory receptor CD40L/CD40 to regulate the priming and functional state of CD8 + T cells.In addition to CD40, the expression of costimulatory receptors on cDC1s, including CD70, CD80/CD86, and CD137L, plays a critical role in CTL activation.Moreover, the cytokines secreted from CD4 + T cells or DCs modulate CD8 + T cells response.DC, dendritic cell; cDC1, classical dendritic cell 1; TCR, T cell receptor; MHC, major histocompatibility complex.
8][149] Besides, the expression of costimulatory receptors, including CD70, CD80/CD86, and CD137L, also plays an important role in CTL activation. 150he activation effects derived from the CD70 located on DCs interacting with the CD27 on CTLs in murine models are critical to delivering help to CD8 + T cells. 151he CD70-CD27 costimulation signal is well known to promote proliferation and differentiation toward effector or memory phenotypes in primed CD8 + T cells. 152,153any experiments also confirmed the requirement of the CD70-CD27 signal in CTL priming by using soluble CD70. 154,155Moreover, blocking the CD70 signal effectively inhibits CD40-mediated CTL priming, 156 highlighting the critical role of the CD70 signal.CD27 costimulatory signals enhance T cell survival through antiapoptosis and prometabolism effects, 157,158 Additionally, CD27 costimulatory signals also increase the expression of IL-12Rβ2, IL-2Rα, and IL-2 in activated CD8 + T cells, which drives the differentiation of CTLs. 151,159,160esides the CD70-CD27 costimulatory signal, CD80/CD86-CD28 costimulation is an additional signal in CTL priming. 161CD28 induces the enhancement of the T cell receptor (TCR) signal, which subsequently primes the CD8 + T cell cycle and changes its metabolism to enhance the expansion of CD8 + T cells. 162,163In antitumor vaccination, the combination of CD27 agonism and PD-1 blockade effectively recapitulates CD4 + T cell help. 164Similarly, PD-1 blockade promotes CD28 costimulatory signals, 165 suggesting that CD27 and CD28 signals play a synergistic role in CD4 + T cell help.

3.1.2
Cytokines from CD4 + T cells or DCs shape the CTL response Besides the upregulation of costimulatory receptors, activated DCs with CD4 + T cell help can also increase the production of cytokines such as type I IFN, IL-12, and IL-15.It has already been proven that type I IFN and IL-12 from activated DCs regulate TNF receptors and certain molecules related to CTL survival and functions. 166In the absence of type I IFN or IL-12, the effector and memory functions of CD8 + T cells are compromised.][169] CD4 + T cells can also deliver help to CD8 + T cells by IL-2 and IL-21 productions.CD4 + T cells-derived IL-2 is one of the most important cytokines as relevant research suggested that it was essential for promoting CD8 + T cells differentiation toward effector cytolytic T cells. 1601][172] Besides IL-2, CD4 + T cells can also provide IL-21. 94,173,174In the absence of IL-21, CTL response were impaired even with enhancement of CD4 + T cell help signals during chronic viral infections. 175Consistently, tumor-infiltrating CD8 + T cells in an IL-21R knockout mouse model exhibited inhibited granzyme B (GZMB) production. 94

3.1.3
The preponderances of helped CD8 + T cells Activated CD8 + T cells differentiate into several subsets, including short-lived effector T cells and long-lived memory T cells. 176,177In antitumor vaccination and virus infection models, the transcriptional differences in CTLs with the help of CD4 + T cells has been identified through RNA-sequencing. 151Accordingly, compared with unhelped CD8 + T cells in mice only treated MHC-Irestricted epitope vaccination, the helped CD8 + T cells in mouse that received antitumor vaccine encoded MHC-II and MHC-I epitopes showed many discrepancies in master proteins about cytotoxicity and cell cycle-the upregulation of T-BET and IRF4, and the downregulation of TCF-7, EOMES, and ID3 were observed. 151Notably, T-BET and EOMES expression levels determine the functional state of CTLs-T-BET tends to drive CD8 + T cells to terminal differentiation-effector phenotypes, 178 while EOMES expression is upregulated during the effector to memory cell transition 179 -and ID3 is required for the generation of memory CD8 + T cells, 180,181 supporting the important role of CD4 + T cell help in formation of polyfunctional CTLs.
In addition, helped CTLs express higher effector molecules, including IFN-γ, TNF, FASL, GZMA, and GZMB. 151Moreover, CTLs without the help from CD4 + T cells upregulate coinhibitory receptors, such as PD-1, LAG-3, and B and T lymphocyte attenuator (BTLA). 151ubsequently, blocking these three coinhibitory receptors in unhelped CD8 + T cells improves tumor control. 151imilar observation was found in a vaccination study that helpless CD8 + T cells exhibited reduced effector function and expressed higher levels of inhibitory receptors. 182hese gene signatures are similar to those of exhausted T cells during chronic viral infections or tumors. 183Therefore, CD4 + T cell help could prevent CD8 + T cells from the exhausted phenotype.
The sequence analysis also demonstrated that helped CD8 + T cells gained a stronger extravasation potential by upregulation of chemokine receptors CXCR4 and CX3CR1, 151 which could bind to CXCL12 and CX3CL1 from tumor cells, thus inducing CTLs infiltration into TME to exert antitumor effects. 184,185The decreased density of infiltrating CD8 + T cells by CXCR4 and CX3CR1 inhibitors also supported this result. 151Except for chemokines, an increased expression of matrix metalloproteinases and a decreased expression of its inhibitory protein TIMIP2 were also found in helped CD8 + T cells, 151 highlighting the important role of CD4 + T cells in promoting the penetration of CD8 + T cells into effector tissues.
As for memory CTLs, activated CD8 + T cells without help upregulated the expression of TRAIL, which mediated apoptosis during the secondary expansion of memory CTLs to restrict their proliferation. 186,187In addition, helped CTLs expressed higher level of inhibitory apoptosis regulator BCL-XL to amplify their survival and proliferation. 168

Interaction of CD4 + T cells with macrophages, NK cells and B cells promotes the antitumor response
In murine models of MHC-II-negative plasmacytoma, IFN-γ secreted by CD4 + T cells can induce macrophage activation and M1-polarization to prevent tumor progression (Figure 3A). 12M1-like macrophages upregulate inducible nitric oxide synthetase (iNOS) and thereby subsequently increase tumor apoptosis triggered by nitric oxide (NO). 188In contrast with M1 macrophages, M2 macrophages activated by IL-4 and IL-13 mainly from Th2 cells 189,190 contribute to increasing the production of IL-10 and arginase, resulting in the downregulation of NO production and restriction of the pathogen clearance ability. 191,192Similarly, CD4 + T cell-derived IFN-γ mediates an increase in tumoricidal effectors, monocytemacrophages, which secrete NO to induce remote inflammatory cell death in IFN-unresponsive, MHC-deficient tumor cells. 193econd, IL-2 produced by CD4 + T cells has been proved to associated with the maintenance and the activation of NK cells, [194][195][196] which could present antitumor effects through direct killing functions in murine models and clinical patients. 197,198dditionally, it has been reported that CD4 + T cells play a vital role in the induction of humoral responses (Figure 3B).][218] Similarly, ICOS plays an important role in the formation of a complete humoral response. 219In addition to these costimulatory signaling, Tfh-derived IL-21 also regulates GC B cell differentiation. 95,215

CD4 + T cells in antivessel formation
CD4 + T cells have been proven to inhibit tumor growth by inhibiting tumor blood vessel formation and maintenance (Figure 3C).According to some early studies, IFN-γ can influence non-bone marrow-derived cells to limit the progression of tumors by inhibiting the blood vessel formation. 220,221Even in tumor cells lacking of the expression of IFN-γ receptors, IFN-γ still helps prevent tumor growth. 220,222In light of IFN-γ-deficient model and the phenomena observed from TNF-deficient model using intravital microscopy, IFN-γ induced vascular degeneration in the process of angiogenesis, inducing an ischemic state in the tumor tissue, while TNF-α causes vascular rupture in the tumor tissue and promotes the infiltration of circulating cells into the TME when destroying blood vessels. 26Therefore, IFN-γ and TNF-α secreted by CD4 + T cells play a synergistic role in the inhibition of angiogenesis in tumor tissue by cutting off the blood supply to solid tumors to reduce tumor survival and metastasis.

Cytotoxic CD4 + T cells-mediated antitumor effects
It is generally believed that the killing effects observed in antitumor immunity are mainly attributed to CTLs, while CD4 + T cells provide help and regulatory effects.4][225][226][227][228][229][230][231][232] In a murine virus infection model, CD4 + CTLs are distinguished by the expression of GZMB and their generation are inhibited by the Tfh-related-transcription factors BCL-6 and TCF-1. 233n addition, CD4 + CTLs lose the expression of THPOK and gain the expression of T-BET and RUNX3 in intraepithelial lymphocytes, 229,232 whereas cytotoxic T-cell-like CD4 + T cells express EOMES in experimental autoimmune encephalomyelitis. 234According to another study on staphylococcal enterotoxin A, CD134 and CD137 costimulation programs the differentiation of cytotoxic Th1 cells, and Gzmb expression in cytotoxic Th1 cells depends on Eomes. 235Moreover, it has reported that CRTAM promotes CD4 + CTL differentiation by inducing the expression of Eomes and cytotoxic genes. 224Furthermore, the expression of Blimp-1 greatly influences the cytotoxic function of CD4 + CTLs during influenza viral infections. 236imilarly, in a murine tumor model, CD4 + T cells can also be differentiated into cytotoxic CD4 + T cells with the secreting of IFN-γ, granzymes, and perforin (Figure 3D). 14,237Moreover, tumor regression mediated by cytotoxic CD4 + T cells is dependent on MHC-IIrestricted manner, which could be enhanced by anti-CTLA-4 antibody. 2380][241][242] According to the results of single-cell sequencing in human bladder cancer, cytotoxic CD4 + T cells are also polyfunctional due to the expression of GZMA, GZMB, GZMK, PRF1, GNLY, and NKG7. 242Such cytotoxic CD4 + T cells directly kill autologous tumors in a MHC-II-dependent manner in response to immunotherapy. 242However, the function of cytotoxic CD4 + T cells could be diminished by autologous Tregs. 242,243Interestingly, cytotoxic activity is partly dependent on SLAMF-7 and enhanced by the combined application of SLAMF-7 agonist. 241In general, the killing function of cytotoxic CD4 + T cells depends on MHC-II and the expression of cytolytic effector molecules.
The expression of transcription factors in the cytotoxic CD4 + T cell subset in TME is various according to the type of tumors.In a melanoma model treated with adoptive cell transfer (ACT), Th1 cytotoxic T cells differentiated from naïve CD4 + T cells secreted granzyme B, perforin and IFN-γ, and expressed the transcription factor T-bet. 237 In contrast, in adoptive transfer model bearing melanoma, Gzmb + CD4 + T cells generated under the additional use of anti-OX40 agonists (OX86) and cyclophosphamide (CTX) were also Eomes + .Compared with Eomes knockdown antigen-specific cells, this combined treatment enhanced the killing function of CD4 + CTLs according to the required expression of Eomes. 2446][247] Notably, the robust transcriptional map of CD4 + CTL generated from single-cell RNA sequencing in patients with melanoma consistently indicates that, compared with Th cells, cytotoxic CD4 + T cells enrich the expression of RUNX3, unlike T-BET, EOMES and BLIMP-1. 241ith the IL-12 inducing production of granzymes and perforin through the STAT4 pathway, Th1 cells acquire cytotoxic functions, 224,248 while CD4 + CTLs can also be generated from various cells, such as Th0, Th1, and Th2, under the action of IL-2 and IL-12 in certain conditions.Among these, IL-2 plays an important role in inducing the differentiation into CD4 + CTLs with improved cytotoxic functions, especially Th0. 224,248

CD+ T CELL ROLES IN PROTUMOR AND PROCHRONIC VIRAL RESPONSES
In addition to antitumor and antichronic viral responses of CD4 + T cells, CD4 + T cells also play a role in protumor and prochronic viral responses.The subpopulation of CD4 + T cells, Treg cells, can mediate an immunosuppression to inhibit effector T cells function, thereby inducing tumor progression and chronic viral persistence.Moreover, exhausted CD4 + T cells exhibit reduced effec-tor function and proliferation and increased expression of inhibitory receptors, which can promote protumor and prochronic viral responses.

Immunosuppression mediated by Treg cells
5][116][117] According to a transcriptome analysis based on human cancers, tumorinfiltrating Treg cells also upregulate the expression of some immunosuppressive molecules, such as LAG-3, TIM-3, T cell immunoreceptor with Ig and ITIM domains (TIGIT), CTLA-4, 254,255 and some molecules involved in activation, including ICOS, CD137, OX40, and GITR. 256n addition, TCF-1-deficient Treg cells could significantly inhibit T cell proliferation and cytotoxicity to promote tumor growth.In colorectal cancer patients, tumorinfiltrating Treg cells expressed lower levels of TCF-1 compared with those in the blood. 2579][260][261][262] Notably, two subsets of FOXP3 + T cells are associated with different outcomes in colorectal cancer.The FOXP3 hi subset is primarily immunosuppressive cells associated with poor prognosis, whereas the FOXP3 lo subset is considered non-Treg cells, which mainly produce inflammatory cytokines leading to better prognosis. 263

Immunosuppression mediated by Treg cells in tumors
The immunosuppressive effect induced by Treg cells was first proposed in studies on tumor rejection by using anti-CD25 to delete CD25 + CD4 + Treg cells. 264,265In TME, Treg cells induce immunosuppressive effects, which predominantly maintained by TCR signaling 266 to inhibit tumor regression by diverse pathways 267,268 In addition to TCR signaling, CD25 can induce STAT5 activation to maintain the suppressive function of Treg cells.CD25, also known as IL-2Rα, cooperating with IL-2R β and γ chains form high-affinity IL-2R complex to competitively bind to IL-2.The competitive depletion of IL-2 prevents further activation of effector T cells and inhibits maintenance of the T cell pool as a consequence of the weak immune response. 2692][123] Another study demonstrated that the trans-endocytosis of CD80/CD86 occurred between APCs and Treg cells when interacting with CTLA-4, leading to the downregulation of CD80/CD86. 122In addition, the interaction of CTLA-4 with CD80/CD86 promotes the production of indoleamine 2,3-dioxygenase (IDO), which is an essential enzyme that degrades tryptophan and subsequently promotes a lack of tryptophan in the TME, resulting in the inhibition of T cell proliferation. 270,271In a mouse model, CTLA-4-deficiency inhibited the immunosuppressive effects of Treg cells, specifically, the Tregmediated downregulation of CD80/CD86 expression on DCs, and enhanced the antitumor response. 1233][274] In addition, the clinical efficacy of the monoclonal antibody ipilimumab (anti-CTLA-4) in patients with clinical melanoma is associated with selective depletion of Treg cells based on the FcγR-dependent mechanism and an increase in the tumor-infiltrating CD8 + T/Treg cell and Teff/Treg ratios. 275[278]

Immunosuppression mediated by Treg cells during chronic viral infection
CD4 + CD25 + Treg cells increase in HBV-infected patients during chronic HBV infection [279][280][281] and mediate decreased proliferation of peripheral blood mononuclear cells (PBMC). 279,281Furthermore, the depletion of CD4 + CD25 + Treg cells can elevate HBV-specific T cells responses. 279,280A decline of PBMC proliferation and IFN-γ production in HBV individuals are observed by restoring depleted CD4 + CD25 + Treg cells. 279Notably, viral load is correlated with the frequency of circulating CD4 + CD25 + Treg cells. 281,282Moreover, Treg cells can mediate an immunosuppressive effect and a defect of immune response to HBV virus. 279n addition, at the initial time of HCV infection, the Foxp3 expression and Treg cells immunosuppression are similar in patients with resolved HCV infection spontaneously or persistence HCV infection. 283However, during HCV persistence, the upregulation of Treg cells has been observed, which directly inhibits HCV-specific CD8 + T cell responses 284 by curtailing the proliferation and perforin expression. 285Similarly, one study also demonstrated that CD4 + CD25 + Treg cells attenuated antigen-specific prolif-eration and IFN-γ production in antigen-specific CD8 + T cells during persistent HCV infection. 286Furthermore, the depletion of CD4 + CD25 + Treg cells mediates an increase in the peptide-specific expansion and the number of IFN-γ + CD8 + T cells 285 during persistent HCV virus infection.However, the impairment induced by CD4 + CD25 + Treg cells is not in antigen-specific manner or limited to HCV-specific CD8 + T cells, but also applies to influenza virus-specific CD8 + T cells. 286In addition to the suppression of antigen-specific proliferation and IFN-γ production in HCV-specific T cells, CD4 + CD25 + Treg cells can impair activation-induced cell death mediated by HCV-specific T cells. 287

CD4 + T cell exhaustion
Owing to continued antigen stimulation, CD8 + T cells become exhausted in tumors and chronic viral infections.Similar to the CD8 + T cells, CD4 + T cells also exhibit an exhaustion status in tumors and chronic viral infections.Meanwhile, exhausted CD4 + T cells exhibit reduced effector function and proliferation, altered transcriptional expression, and increased expression of inhibitory receptors, such as CTLA-4, PD-1, LAG-3, and CD160 (Figure 4).

Exhausted CD4 + T cells in tumors
9][290] In a melanoma tumor model, the adoptive transfer of tumor antigen-specific CD4 + T cells can prevent tumor progression.However, some of the mice experience tumor relapse. 291,292Tumor-infiltrating CD4 + T cells in recurrent tumors express higher levels of inhibitory receptors and have a lower capacity of cytokine production. 292Compared with peripheral blood derived CD4 + T cells 293,294 and nonmalignant infiltrating CD4 + T cells, [295][296][297] tumor-infiltrating CD4 + T cells express high levels of inhibitory receptors. 298This indicates that continuous antigen stimulation can induce exhaustion of CD4 + T cells.In addition, the expression levels of inhibitory receptors on CD4 + T cells seem to be associated with tumor progression and clinical stage. 299,3002][303][304] In addition to inhibitory receptors, CD4 + T cells in TME exhibited poor effector function in a murine melanoma model. 305In human patients, CD4 + T cells in tumors also exhibit reduced proliferation and cytokine production. 306,307Nevertheless, ICB can partially reinvigorate the function of CD4 + T cells. 295,299,307,308In addition, the combination of PD-L1 and LAG-3 blockade downregulates the expression levels of inhibitory receptors of CD4 + T cells. 292,309CF-1 is a key transcription factor for the progenitor exhausted CD8 + T cells. 310In addition to TCF-1, Wherry et al. defined SLAMF6 as an important marker of the progenitor exhausted CD8 + T cells. 3113][314] Compared with CD4 + T cells in spleen, tumor-infiltrating CD4 + T cells lost expression levels of TCF-1 and SLAMF6 in a melanoma model, indicating that tumor-infiltrating CD4 + T cells seem to be a transitory or terminal exhausted T cells. 315When treated with a PD-L1 blockade, tumor-infiltrating CD4 + T cells upregulate TCF-1 and downregulate TIM-3 and LAG-3. 315D39 has been reported as a marker of CD8 + T cell exhaustion, with CD39 high CD8 + T cells showing reduced capacity of cytokine production.316 Tumor-infiltrating CD4 + T cells also express CD39, which express higher levels of PD-1 and produce fewer cytokines.317 After PD-1 blockade, CD4 + T cells have been reported to produce more cytokines.317 Moreover, PD-1 blockade enhances DC maturation and CD8 + T cells proliferation.317 TOX can drive the epigenetic programming of exhausted CD8 + T cells [318][319][320] with the highest expression being exhibited by terminal exhaustion T cells compared with other exhaustion subsets.321 Similar to tumor-infiltrating CD8 + T cells, those CD39 + CD4 + T cells also express TOX.317

4.2.2
Exhausted CD4 + T cells in chronic viral infection CD4 + T cells are unessential for controlling acute viral infections, while they play an important role in the elimination of chronic viral infection due to their roles of helping CD8 + T cells priming and sustaining their function.Similar to CD8 + T cells, CD4 + T cells become exhausted due to continued antigen stimulation during chronic viral infection.Nonetheless, exhausted CD4 + T cells received less attention and was less known about their mechanism of action during chronic viral infection.Compared with LCMV Armstrong infection, during the early stage post LCMV clone-13 infection, continued viral antigen stimulation mediates the functional inactivation of virus-specific CD4 + T cells with losing the ability of producing effector cytokines, such as IFN-γ and TNFα. 143Despite the decline in IFN-γ, the IFN signaling is sustained in virus-specific T cells, 322,323 which in turn modulates an increase in the ISG signature.In addition, IL-21, which is critical for CD8 + T cells and B cells, increases during chronic viral infections as compared with acute viral infections. 175,324Moreover, IL-10, which suppresses T cells function, also drastically upregulates during LCMV clone-13 infection 325,326 and HIV infections. 327,328n acute HCV infection, the vigorous proliferation of virus-specific CD4 + T cells in blood is correlated with the elimination of HCV virus. 329Moreover, multiepitopespecific CD4 + T cells are detected at an early stage of HCV infection, suggesting a broad response of CD4 + T cells in HCV-infected individuals. 329The breadth of the response and the repertoire of targeted epitopes are not significantly different between acute spontaneously resolving (AR) individuals and acute chronically evolving (AC) HCV-individuals. 329Nevertheless, consistent with the exhausted CD8 + T cells, the proliferative capability of exhausted CD4 + T cells is impaired during chronic viral infection. 330,331The early reduction in proliferative capability of HCV-specific CD4 + T cells is correlated with persistent viremia. 329Meanwhile, virus-specific CD4 + T cells progressively decreased due to persistent viral antigens. 329he alterations observed in exhausted CD4 + T cells include not only the loss of poly-functionality and proliferative capability, but also the expression of multiple inhibitory ligands.3][334][335] In addition, PD-1 expression in HIV-specific T cells is significantly higher in the lymph node than in the blood. 332onsistent with PD-1 expression, the expression level of CTLA-4 also increases in virus-specific CD4 + T cells during HIV infection and is positively correlated with disease progression. 334,336Moreover, Tim-3 is also coexpressed with PD-1 and CTLA-4 in virus-specific CD4 + T cells during HIV infection. 337In addition to these inhibitory molecules, virus-specific CD4 + T cells also coexpress other inhibitory ligands during LCMV clone-13 infection, including CD160 and LAG-3. 338Similarly, CD4 + T cells express high level of PD-1 and LAG-3 during chronic HBV infection. 3391][342] At the early time of viral infection, CXCR5 + SMATA cells equally upregulate in both acute infection and chronic infection. 341Strikingly, due to persistent antigen stimulation during chronic infection, CXCR5 + SMATA cells, which are referred to as Tfh cells, dramatically increase. 341In contrast, the frequency of CXCR5 + SMATA cells are comparable at different periods of acute viral infection. 341Sustained antigen or TCR stimulation maintains the expression of Tfh-associated transcription factors, such as CXCR5 expression. 341,343oreover, master transcription factors in virus-specific Tfh cells also increase during chronic infection, including ICOS and Bcl-6. 341Heterogeneous transcription factor profiles of exhausted CD4 + T cells confirm a loss of Th1 cells and an enrichment of Tfh cells during chronic viral infection. 340Similar to chronic LCMV infection, Tfh cells also increase in HIV-infected individuals. 344Moreover, an accumulated population of Tfh cells exhibit an uncomparable transcriptional profiles in SIV-infected tissues compared with SIV-negative tissues.
Tfh cells are critical for B cells to produce classswitched and high-affinity antibodies.Neutralizing antibodies are generated in acute infection; however, they are impaired in Tfh cells during chronic viral infection. 345n contrast, non-neutralizing antibodies are enhanced 345 during chronic infection.Furthermore, polyclonal hypergammaglobulinemia is exhibited in chronic virus-infected mice 345,346 and is associated with the expression level of BCL-6 in HIV-Tfh cells. 346During chronic hepatitis C infection, vaccine responses toward hepatitis B virus are impaired and CD4 + T cell responses are downregulated in vaccine nonresponders compared with those in vaccine responders, indicating the defect of Tfh-B cells interaction and a poor ability of Tfh cells to help B cells. 347Similarly, Tfh cells have an inability to relay help to B cells in HIVinfected individuals. 3449][350] The HIV viral load is correlated with the percentage of Tfh cells. 350Moreover, Tfh cells are a restrict compartment for SIV virus and highly reside in B cell follicles of lymph nodes. 350SIV-specific CD8 + T cells can only mediate the clearance of SIV virus at extra-follicular sites.Hence, SIV virus cannot be cleared in virus-infected Tfh cells, 348 suggesting that Tfh cells can prevent SIV virus from being eliminated by SIV-specific CD8 + T cells.

CD4 + T CELL-BASED IMMUNOTHERAPIES
Immunotherapies, including ACT therapy, vaccines, and ICB, have revolutionized cancer and chronic viral treatments.Most of these immunotherapies are mainly focused on CD8 + T cells; however, CD4 + T cells-based immunotherapies recently receive more attention in tumors and chronic viral infections due to the crucial roles of CD4 + T cells.Notably, some of the CD4 + T cells-based immunotherapies have already been approved, such as CTLA-4.All of the clinical trials of the immunotherapies based on CD4 + T cells are listed in Table 1.

Adoptive cell transfer
ACT therapy mainly involves the use of natural tumorreactive lymphocytes, genetically engineered lymphocytes expressing chimeric antigen receptors (CARs) or TCRs (Figure 5A).Most of the ACT therapies focus on CD8 + T cells instead of CD4 + T cells due to the robust effector function of CD8 + T cells.However, the transfer of autologous NY-ESO-1-specific CD4 + T cells can induce a durable clinical remission in patients with metastatic melanoma.Moreover, ACT with approximately 25% mutation-specific Th1 cells was shown to mediate tumor regression in patients with metastatic epithelial cancer. 351Upon tumor progression, the patients were re-transferred with >95% pure mutation-specific Th1 cells, which effectively sup-pressed the tumor volume, 351 indicating that CD4 + T cells can also be harnessed for ACT treatment.During the CAR-T therapy for patients with myeloma, a higher CD4/CD8 ratio is associated with a better clinical response. 374,375oreover, the administration of CD4 + M28z CAR-T cells alone for mesothelioma is efficacious and contributes to tumor suppression and the administration of CD4 + 19z1 + T cells alone for B cell tumors could prolong the survival of tumor-bearing mice. 376,377In accordance with previous studies, the adoptive treatment with MHC-II-restricted and MAGE-A3-specific CAR-T cells is efficacious to generate tumor eradication, which highlights the importance of CD4 + T cells in ACT therapy. 378wing to continuous antigen stimulation, the transferred autologous CD8 + T cells, CD8 + CAR-T cells, 352,[379][380] or CD8 + TCR-T cells 381,382 become rapidly exhausted and loss the capacity of effector function.One study reported that, in contrast to CD8 + CAR-T cells, glioblastoma-targeted CD4 + CAR-T cells could maintain the T cell pool and retain its effector function. 352Consistent with the report, CD4 + CAR-T cells can sustain efficacy in vivo compared to CD8 + CAR-T cells. 382Meanwhile, CD4 + CAR-T cells also exhibit equivalent cytotoxicity with CD8 + CAR-T cells. 382Moreover, administration of the CD4-targeted lentiviral vector to generate CAR-T cells induces a better killing function than administration of the CD8-targetd lentiviral vector. 383Surprisingly, CD4 + CAR-T cells are slightly more polyfunctional than CD8 + CAR-T cells and have a highly mixed Th1/Th2 function. 47oreover, TCR engineered BRAFV600E-specific CD4 + T cells have been reported to enhance the CD8 + T cell response in patients with melanoma and induce an antitumor effect. 384 Furthermore, T-bet can improve the antitumor effect of CD4 + CAR-T cells against B7H6-expressing tumor cells by upregulating the secretion of cytokines and cytotoxicity.Recently, long-persisting CD4 + CD19 + CAR-T cells have been reported to emerge in patients with chronic lymphocytic leukemia who achieved complete remission. 355,356uch long-persisting CD4 + CD19 + CAR-T cells display cytotoxicity, activation, and proliferation. 355

Vaccines
To exploit the cytotoxic capacity of CD8 + T cells, most of the vaccines have been designed using MHC-I-restricted epitopes. 386,387However, some of the CD8 + T cell-based vaccine strategies do not induce favorable clinical outcomes.For example, a CD8 + T cell-based vaccine strategy without CD4 + T cell help cannot induce the formation of cytotoxicity in CD8 + T cells. 77Besides, a synthetic longpeptide (SLP) vaccine predicted to bind MHC class I raised CD4 + T cell responses. 388Similarly, despite the absence of MHC-II binding prediction, a synthetic RNA mutanome vaccine for MHC-I-restricted prediction has also been reported to elicit CD4 + T cell responses. 389Likewise, neoantigen vaccines with MHC-I binding affinity also yield a CD4 + T cell response, 390,391 suggesting that the generation of polyfunctional CD8 + T cell responses is associated with CD4 + T cell help.In addition, continuous MHC-I-restricted epitope stimulation promotes CD8 + T cell exhaustion. 392,393Therefore, MHC-II-restricted epitopebased vaccine strategy has been utilized (Figure 5B) and the clinical trials of these vaccines are listed in Table 1.
In patients with prostate cancer, a SLP vaccine containing CD4 + T cell helper epitopes can induce both CD4 + and CD8 + T cell responses. 357,358Similar observations have been reported for a SLP vaccine with the inclusion of a helper epitope in patients with human papillomavirus (HPV)-induced intraepithelial neoplasia. 394,359In patients with pancreatic cancer, a mutated Ras-SLP was aimed to predominately activate CD4 + T cells can be synergized with GM-CSF and mediate a robust CD4 + T cell response to prolong survival, 360,395 underlining the crucial role of MHC-II-restricted epitopes in immunotherapies.Human telomerase reverse transcriptase (h-TERT) is expressed in multiple types of cancer, including nonsmall-cell lung cancer (NSCLC).97] Concomitantly, the vaccines based on HLA-II binding h-TERT derived epitopes referred as universal cancer peptides (UCP) elicit Th1 and CD8 + T cell responses and promote the infiltration of h-TERT-specific CD8 + T cells, 398,399 confirming that MHC-II-restricted epitopebased vaccines can mediate potent antitumor effects.This UCP-based vaccine was evaluated on patients with glioblastoma (NCT04280848) and metastatic NSCLC 363 in clinical trials.UCP-based vaccine combined with ICB was also evaluated in individuals with HPV positive cancers 369 and NSCLC (NCT04263051).Moreover, MHC-II epitopes can shape antitumor immunity and synergize with immunotherapy. 400,401In our study, we found that the MHC-II-restricted epitope-based heterologous primeboost vaccination potentiates antitumor immunity and PD-1/PD-L1 immunotherapy. 402Besides, a heterologous prime-boost immunization based on MHC-II-restricted epitope selectively induces virus-specific CD4 + T cell responses and control chronic viral infection. 403To attempt to take advantage of both CD8 + T cells and CD4 + T cells, MHC-I and MHC-II-based vaccination was carried out and displayed strong immunogenicity. 364,365Since DCs delivery CD4 + T cell help to CD8 + T cells, several studies have initiated DC-based vaccine pulsed by the HER2 MHC-II epitopes 366,[404][405] and these vaccine strategies mediate tumor elimination. 366,405

Immune checkpoint blockade
7][408] Likewise, the positive clinical outcome reflects an enhanced percentage of CD4 + T cells in patients treated with ipilimumab, 409 indicating that CD4 + T cells play a crucial role in the ICB therapies. 410In addition, the repertoire of systemic CD4 + T cells prior to immunotherapy correlates with the antitumor efficacy of CTLA-4 or PD-1 blockade therapy. 411Moreover, CD4 + CD62L low T cells in the blood prior to immunotherapy are correlated with the response to PD-1 blockade, 412 highlighting the critical contribution of CD4 + T cells.The efficacy of CTLA-4 blockade is due to the tumor-infiltrating Treg cell depletion or reduction. 273,274Interestingly, the combination of CTLA-4 and PD-1 blockade increases the percentage of Th1-like CD4 + T cells. 413In addition, CTLA-4 blockade mediates ICOS + (or ICOS hi ) Th1-like CD4 + T cell expansion. 31,414Furthermore, CD44 + PD-1 − CD127 low CD4 + T cells, referred as unexhausted cells, significantly increase in responders treated with ICB, 415 demonstrating that unexhausted CD4 + T cells expand to restrain tumor progression in response to ICB treatment.In addition, tumor-specific cytotoxic CD4 + T cells express lytic granules induced by Eomes after ipilimumab treatment in patients with advanced melanoma. 367D-1/PD-L1 blockade can restore the function and proliferation of HIV-specific CD4 + T cells.[332][333][334][335] Moreover, PD-1 pathway blockade can enhance HIV-specific immunoglobulin production, which restores Tfh help to B cells during HIV infection.344 In humanized mice, blocking the PD-1 pathway induces a progressive reduction in HIV viral load and a significant increase in the percentage of CD4 + T cells compared with untreated mice.416 Furthermore, PD-1 blockade enhances HIV-1-specific T cell function.416

Other therapies
In addition to the aforementioned therapies, various other therapies based on CD4 + T cells have been developed (Figure 5D).In patients with prostate cancer in situ, immune checkpoint inhibitors increase the number of tumor-infiltrating Th1 cells; however, in patients with bone metastasis of prostate tumor, immune checkpoint inhibitors increase Th17 subsets but not Th1 due to the presence of TGF-β. 368Therefore, the combination of anti-CTLA-4 and anti-TGF-β treatment allows Th1 development and augments the efficacy of ICB. 368Meanwhile, it has also been reported that blocking TGF-β signaling, such as TGF-β receptor II (TGF-βRII), in CD4 + T cells mediates tumor regression by promoting the remodeling of the blood vasculature, which means a mature and organized tumor vasculature. 417,418Notably, CD27 agonism has been reported to mimic CD4 + T cell help acting on CD8 + T cells to enhance CTL responses. 164Moreover, CD27 antibody has been proven to be safe in clinical trials. 370Interestingly, the combination of CD27 agonism and PD-1 blockade recapitulates CD4 + T cell help and improves the efficacy of the immunotherapy, suggesting that CTLs primed by CD27 agonism may also experience immune suppression induced by the PD-1/PD-L1 pathway in tumors, which can be reversed by PD-1 blockade. 164 In metastatic breast cancer, CD25-blocking monoclonal antibody daclizumab can mediate reduction and dysfunction of CD25 hi CD45RA neg Treg cells and reinvigorate the capability to secrete IFN-γ.The combination of daclizumab and cancer vaccine can induce a robust CD8 + and CD4 + T cell priming and boosting and a decline of Treg cells. 420The p110δ inactivation in Tregs cells can mediate elimination of tumor cells and reinvigorate CD8 + cytotoxic T cells in multiple murine tumor models. 421CCR4 + CD45RA − FOXP3 hi CD4 + Treg cells (effector Treg cells, eTreg cells) are predominately among tumor-infiltrating Treg cells in melanoma.The treatment of anti-CCR4 mAb mediates a reduction of eTreg cells and upregulation of antigen-specific CD8 + T cells response. 422uring HIV infection, IL-10 is upregulated and impairs CD4 + T cells activation. 327,328Blockade of the IL-10/IL-10 receptor pathway mediates viral clearance and functional improvement in T cells. 327,328Furthermore, IL-10 blockade can also significantly enhance the proliferation of HIV-specific CD4 + T cells and elevate the secretion of IFN-γ and IL-2. 327PD-L1 blockade restores IFN-γ, IL-2, and IL-13 production, while blockade of IL-10 receptor pathway remarkably enhances IFN-γ secretion. 423The combination of PD-L1 blockade and IL-10Rα blockade mediates a dramatic increase in IFN-γ and an upregulation of IL-2, and IL-13 production in HIV-specific CD4 T cells.

A U T H O R C O N T R I B U T I O N
L. X., J. F., J. Y., W. Z., and Z. H. conceived, edited, and wrote this manuscript.H. W. and L. Y. revised this manuscript.All authors have read and approved the final manuscript.

A C K N O W L E D G M E N T S
This work was supported by grants from the National Natural Science Foundation of China (82072723).

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare they have no conflicts of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
Not applicable.

E T H I C S S TAT E M E N T
The author declare that ethics approval was not needed for this study.

F I G U R E 1
Classification and ontogeny of CD4 + T cells.Cytokines and transcription factors promote differentiation of naïve CD4 + T cells into several sub-populations.According to the differences in expressions of transcriptional factors and functions, they are classified into Th1, Th2, Th9, Th17, Th22, Tfh, regulatory T cell (Treg), and cytotoxic CD4 + T cells.Specifically, the phenotype and killing function of cytotoxic CD4 + T cells in tumor microenvironment have been distinguished and represented by the expression of granzymes and/or perforin.CTL, cytotoxic T lymphocyte; Th1, T helper 1 cell; Th2, T helper 2 cell; Th9, T helper 9 cell; Th17, T helper 17 cell; Th22, T helper 22 cell; Tfh, follicular helper T cell; Treg, regulatory T cell; IFN, interferon; TNF, tumor necrosis factor; TGF-β, transforming growth factor β.

F I G U R E 3
The indirect and direct functions of CD4 + T cells in antitumor and antichronic viral responses.(A-C) The indirect functions of CD4 + T cells in antitumor and antichronic viral response.(D) The direct cytotoxic function of CD4 + T cells.(A) CD4 + T cells can produce IFN-γ and then upregulate the expression of iNOS in macrophages.iNOS in macrophages promotes the production of NO in TME and subsequently induces tumor cell apoptosis.CD4 + T cells can also help NK cells activation and maintenance by IL-2.iNOS, inducible nitric oxide synthetase; NO, nitric oxide.(B) CD4 + T cell can interact with B cell by CD40L-CD40 interaction to mediate humoral immune response, such as the generation of memory B cells, the formation of GC and the generation of affinity-matured and class-switched plasma cells.In addition, Tfh-derived IL-21 also regulates GC B cell differentiation.(C) CD4 + T cells secrete IFN-γ and TNF-α to help reject tumors through damaging the formation of the vessel systems in tumor tissue.(D) Cytotoxic CD4 + T cells secrete IFN-γ, granzymes, and perforin to mediate the direct tumor killing function.

F I G U R E 4
Treg cells and exhausted CD4 + T cells in protumor and prochronical viral infection.(A) Treg cells can mediate immunosuppressive functions through various ways, including CD25, CTLA-4, IL-10, TGF-β, and IL-35.CD25 cooperating with IL-2R β and γ chains can competitively binding to IL-2 to inhibit activation and maintenance of effector T cells.In addition, CTLA-4 on Treg cells contributes to decreasing the CD28-CD80/CD86 signal on DCs and increasing the secretion of IDO from DCs, inducing a lack of tryptophan in TME and subsequently inhibiting T cell proliferation.IDO, indoleamine 2,3-dioxygenase; IL-2R, IL-2 receptor.(B) Exhausted CD4 + T cells exhibit upregulation of TOX and inhibitory receptors, including PD-1, CTLA-4, LAG-3, and CD160, while lose the expression of SLAMF-6 and TCF-1.Additionally, exhausted CD4 + T cells mediate a reduction of proliferation and function with decreased secretion of IFN-γ and TNF-α.However, IL-21 and IL-10 are significantly upregulated.Furthermore, CD4 + T cells exhibit a loss of Th1 cells and an enrichment of Tfh cells.

F I G U R E 5
Immunotherapies based on CD4 + T cells.(A) Adoptive cell transfer (ACT) therapy includes autologous CD4 + T cells transfer, CD4 + chimeric antigen receptor (CAR) T cell transfer, and engineered CD4 + T cell receptor (TCR) T cell transfer.(B) Vaccines.MHC-II-restricted epitope-based vaccine strategy, MHC-I-restricted epitope and MHC-II-restricted epitope-based vaccine strategy, synthetic long-peptide (SLP) containing helper epitope vaccine strategy, and dendritic cell-based vaccines are available.(C) Immune checkpoint blockade aims to inhibit immune checkpoint receptors axis, including CTLA-4 and PD-1.(D) Other treatments.Considering the important role of cytokines and costimulatory receptors, there are some other strategies, such as anti-TGF-β and CD27 agonism and so on.

6 CONCLUSION
CD8 + T cells have received considerable attention in tumor immunotherapy strategies due to the important role of CTL cells in the killing effect of tumor cells and chronic viruses.In TME and chronic viral infections, the function of CD8 + T cells gradually decreases, and the expression of inhibitory receptors, such as the immune checkpoint of PD-1 and CTLA-4, gradually increases.In advanced stages of exhaustion, CD8 + T cells may even disappear.Additionally, many CD8 + T cell-based immunotherapies, such as ICB, cannot completely reverse the progression of exhaustion.Moreover, when MHC-I epitope-based vaccines are utilized, persistent tumor antigens accelerate the exhaustion of CD8 + T cells.CD4 + T cells also play an important role in antitumor immunity and antichronic viral immunity owing to their auxiliary effects.Helping signals from CD4 + T cells are delivered by DCs to CD8 + T cells.This auxiliary effect acts on CD8 + T cells through costimulatory molecules, cytokines, or chemokines to affect the activation, proliferation, differentiation, and migration of CD8 + T cells.In addition to this auxiliary effect, some of the CD4 + T cells have a direct killing function in the antitumor and antichronic viral responses.Moreover, CD4 + T cells interact with B cells and induce affinity-matured and class-switched plasma cells to regulate antitumor and antichronic viral humoral immunity.On the contrary, Treg cells mediate immunosuppression in the antitumor and antichronic viral activities.Thus, immunotherapies have been developed based on the mechanisms of CD4 + T cells.Specifically, the adoptive transfer of CD4 + T cells or CD4 + CAR T cells can induce tumor regression and a reduction in chronic viral infections, and these cells display both cytotoxicity and polyfunctionality.MHC-II-restricted epitope-based vaccines can also mediate tumor eradication in different types of tumors and prolong survival.Additionally, the CTLA-4 blockade strategy can reverse the immunosuppression effect mediated by Treg cells.Moreover, since DCs play an important role in delivering help signals from CD4 + T cells, DC-based vaccines and the CD27 agonism have recently been exploited to inhibit tumor growth. 373 Clinical trials of the immunotherapies based on CD4 + T cells.
TA B L E 1